1. Chromosomes and DNA Packaging Chapter 5

2. The Problem Human genome (in diploid cells) = 6 x 10 9 bp Human genome (in diploid cells) = 6 x 10 9 bp 6 x 10 9 bp X 0.34 nm/bp = 2.04 x 10 9 nm = 2 m/cell 6 x 10 9 bp X 0.34 nm/bp = 2.04 x 10 9 nm = 2 m/cell Very thin (2.0 nm), extremely fragile Very thin (2.0 nm), extremely fragile Diameter of nucleus = 5-10  m Diameter of nucleus = 5-10  m  DNA must be packaged to protect it, but must still be accessible to allow gene expression and cellular responsiveness  DNA must be packaged to protect it, but must still be accessible to allow gene expression and cellular responsiveness

3. Solution: Chromosomes Single DNA Molecule and associated proteins Single DNA Molecule and associated proteins Karyotype Karyotype Chromatin vs. Chromosomes Chromatin vs. Chromosomes

4. HISTONES Main packaging proteins Main packaging proteins 5 classes: H1, H2A, H2B, H3, H4. 5 classes: H1, H2A, H2B, H3, H4. Rich in Lysine and Arginine Rich in Lysine and Arginine

5. HISTONES NOTE: if histones from different species are added to any eukaryotic DNA sample, chromatin is reconstituted. Implication? NOTE: if histones from different species are added to any eukaryotic DNA sample, chromatin is reconstituted. Implication? Very highly conserved in eukaryotes in both Very highly conserved in eukaryotes in both Structure Structure Function Function Protection Protection Must allow gene activity Must allow gene activity

6. WEAVER: TABLE 13.1 Variability in Histones

7. HISTONES Fairly uniform, some variability Fairly uniform, some variability Why variable if only function is packaging? Why variable if only function is packaging?

8. Variations In Histones How can cells introduce changes in protein structure and thus protein function? How can cells introduce changes in protein structure and thus protein function? Mutations Mutations Post transcriptional modifications—ex alternate splicing Post transcriptional modifications—ex alternate splicing Post translational modifications Post translational modifications Acetylation Acetylation Acetylation Methylation Methylation Ser-Thr O-phosphorylation Ser-Thr O-phosphorylation Ser-Thr O-phosphorylation Ser-Thr O-phosphorylation His N-phosphorylation His N-phosphorylation His N-phosphorylation His N-phosphorylation NOTE: These processes are dynamic. They give the cell another means to regulate gene expression NOTE: These processes are dynamic. They give the cell another means to regulate gene expression

9. DNA is complexed with proteins and organized as chromatin in the interphase nucleus:

10. Orders of chromatin structure from naked DNA to chromatin to fully condensed chromosomes... Fig. 9

11. Beads on a String—10 nm Fiber

12. 10 nm filament; nucleosomes histones (= 1g per g DNA) DNA protein purification H1 H3 H2A H2B H4 Basic (arg, lys); + charges bind to - phosphates on DNA

13. deoxyribonuclease I (DNase I) digestion nucleosomes Conclude: histones in a nucleosome protect 146 nt from DNase I attack. Separate DNA from protein H1 2H3 2H2A 2H2B 2H4 the histone octamer proteins 146 nt fragments DNA

14. 10 nm Fiber A string of nucleosomes is seen under EM as a 10 nm fiber A string of nucleosomes is seen under EM as a 10 nm fiber

15. 30 nm Fiber 30 nm fiber is coil of nucleosomes with 6/turn 30 nm fiber is coil of nucleosomes with 6/turn

16. The 30 nm Fiber (Compacts DNA 7X more)

17. Orders of chromatin structure from naked DNA to chromatin to fully condensed chromosomes... Fig. 9

18. Acid extraction removes histones from meta- phase chromosomes, leaving nonhistones... Low power high power Scaffold protein DNA loops Major non-histone proteins = topoisomerases!

19. Different forms of chromatin show differential gene activity. euchromatin heterochromatin

20. In sperm heads, DNA must be further compressed In sperm heads, DNA must be further compressed Protamines Protamines ~2/3 rd basic amino acids, (mostly Arg) ~2/3 rd basic amino acids, (mostly Arg) Greater degree of compaction associated with: Greater degree of compaction associated with: Fit in DNA Fit in DNA Salt bridges between Arg residues in both the contact helix and adjacent helices Salt bridges between Arg residues in both the contact helix and adjacent helices Protamines

21. Other DNA Associated Proteins Gene expression in a dynamic environment Gene expression in a dynamic environment DNA associated non-histones: DNA associated non-histones: Palindromes Palindromes  -helices  -helices Symmetrical dimers Symmetrical dimers Interaction between R-groups and specific bases Interaction between R-groups and specific bases Role of basic amino acids Role of basic amino acids

22. back

23. Phosphorylation (Ser and Thr) back

24. Histidine: N- phosphorylation How will this affect His’s ability to participate in weak interactions? How will this affect His’s ability to participate in weak interactions? Back

26. Euchromatin (E) vs Heterochromatin (H) E H DNase Being more condensed (tightly packed), heterochromatin is resistant to DNase digestion. Fig. 11

27. Hypothesis: Transcriptionally active DNA (i.e., an active gene) is in euchromatin. Can test with DNase I, which should preferentially digest active genes in a cell... E H nascent transcripts

28. An Experiment: chick red blood cellschick RBC nuclei chromatin extraction total chromatin digest with DNase I for different times… partially digested chromatin; extract undigested DNA denature DNA, “spot” on filter Increasing time of digestion make 2 of these... Fig. 12

29. Radiolabel, denature cloned globin and other gene DNA to use as *probe. Allow *probe DNA to hybridize to complementary sequences on filters… e xpose, develop autoradio- graph... globin gene probe ovalbumin gene probe Fig. 13 Increasing time of digestion DNA from digested RBC chromatin

30. Compared to globin gene DNA, ovalbumin gene DNA in RBC chromatin resists DNase digestion. Globin gene DNA in RBC chromatin must be more accessible to DNase (less condensed, more euchromatic) than ovalbumin DNA. Conclusions:

31. Histones—Degrees of Conservation H4---Only 2 variations ever discovered H4---Only 2 variations ever discovered H3—also highly conserved H3—also highly conserved H2A, H2B---Some variation between tissues and species H2A, H2B---Some variation between tissues and species H1-like histones H1-like histones H1—Varies markedly between tissues and species H1—Varies markedly between tissues and species H1 º --Variable, mostly present in non- replicating cells H1 º --Variable, mostly present in non- replicating cells H5---Extremely variable H5---Extremely variable

33. Post-Translational Modifications

34. 30 nm solenoid fiber 10 nm filament; nucleosomes [salt] Dissection of interphase nuclei... low [salt] extraction